Although adult hematopoietic stem cells (HSCs) are being used routinely in clinical transplantation, the lack of a suitable source of donor cells remains a major limiting factor depriving many patients of this potentially curative treatment. The ultimate goal of the Principal Investigator is to generate HSCs from embryonic stem cells (ESCs) as a novel source of cells suitable for clinical transplantation. We are proposing an intensive research program to study the developmental biology of hematopoiesis from rhesus ESCs, and to establish the rhesus macaque model as a relevant pre-clinical large animal model for the evaluation of the efficacy and safety of ESCderived HSCs. Using methodologies such as co-culturing with mesenchymal stromal cells (MSC) of murine bone marrow origin, non-human primate and human ESCs have been differentiated into primitive hematopoietic cells as defined by surrogate in vitro assays. However, those derived cells have been shown to possess only limited in vivo engraftment potential upon transplantation into NOD/SCID immunodeficient mice. Our underlying hypothesis is that by providing the appropriate extrinsic and intrinsic inductive mechanisms that play critical roles in embryonic hematopoiesis, we can effectively recapitulate those processes in vitro and thus promote the efficient differentiation of rhesus ESCs into HSCs that will be capable of engraftment and long-term hematopoietic reconstitution in vivo. In aim-1 we will generate MSC cells from rhesus ESCs and will test their potential as a hematopoietic supportive microenvironment in coculture experiments for the generation of HSCs from rhesus ESCs. In aim-2 we will test our hypothesis that the over-expression of HOXB4, a transcription factor which plays a key role in embryonic hematopoiesis, in rhesus ESCs via gene transfer, or the exogenous delivery of the HOXB4 protein, can improve the efficiency of their differentiation into HSCs. In aim-3 we will assess the in vivo engraftment potential of hematopoietic cells derived from rhesus ESCs alone or when co-transplanted with rhesus ESC-derived MSCs into NOD/SCID mice as we hypothesize that the co-transplantation of rhesus ESC-derived MSCs would potentially provide an appropriate niche and thus enhance the in vivo engraftment potential of rhesus ESCderived HSCs. The results of these proposed studies would help us to design a rational approach toward moving such cells into actual rhesus transplantation experiments. Embryonic stem cells have the potential to generate an unlimited number of diverse differentiated cells for human therapeutic applications including hematopoietic stem cells. We are proposing to develop a rhesus monkey transplantation model to study the efficacy and safety of such cells as they should be carefully tested in appropriate pre-clinical animal models before being moved into human clinical applications. (End of Abstract)